Abstract
Inability to culture the phloem-restricted alpha-proteobacterium “Candidatus Liberibacter asiaticus” (“Ca. L. asiaticus”) or the closely related species (“Candidatus Liberibacter americanus” and “Candidatus Liberibacter africanus”) that are associated with Huanglongbing (HLB) hampers the development of effective long-term control strategies for this devastating disease. Here we report successful establishment and long-term maintenance of host-free “Ca. L. asiaticus” cultures, with the bacterium growing within cultured biofilms derived from infected citrus tissue. The biofilms were grown in a newly designed growth medium under specific conditions. The initial biofilm-based culture has been successfully maintained for over two years and has undergone over a dozen subcultures. Multiple independent cultures have been established and maintained in a biofilm reactor system, opening the door to the development of pure culture of “Ca. L. asiaticus” and the use of genetics-based methods to understand and mitigate the spread of HLB.
Highlights
Huanglongbing (HLB), known as citrus greening disease, is a threat to worldwide citrus production, a multibillion dollar industry
Diagnostic tests have consistently shown that citrus trees displaying symptoms of HLB in the USA are infected with “Candidatus Liberibacter asiaticus” (“Ca. L. asiaticus”), a phloem-restricted alpha-proteobacterium that is spread by the Asian citrus psyllid (Diaphorina citri, ACP) [1,2,3,4]
We used a new medium with a highstrength buffer along with nutrients including amino acids, trace minerals and vitamins. We showed that this combined system is able to support long-term growth and repeated sub-culturing of “Ca. L. asiaticus” together with other microorganisms in a mixed-community biofilm
Summary
Huanglongbing (HLB), known as citrus greening disease, is a threat to worldwide citrus production, a multibillion dollar industry. The biofilm culture of “Ca. L. asiaticus” was initiated from citrus plant extracts.
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